Biology subjects

Cole, B. A.

Publications and source records attributed to Cole, B. A..

6 recordsLinked to original sources

bioRxiv2026

Structural mechanism of TRPC3 inhibition by a potent and selective antagonist

The transient receptor channel canonical family member 3 (TRPC3) is a non-selective, calcium-permeable cation channel within the larger TRP superfamily. TRPC3 is expressed in both excitable and non-excitable cells, where it integrates multiple signaling pathways and is directly activated by binding of diacylglycerol following activation of G-protein coupled receptors. Increased TRPC3 signaling is implicated in several diseases, highlighting TRPC3 as a potential therapeutic target. However, the development of TRPC3 inhibitors with suitable potency and selectivity has been challenging due to the channels high structural and sequence homology with related family members, particularly TRPC6. Here, we present a novel, selective and highly potent TRPC3 antagonist (GSK2820986A) featuring an anilino-thiazole pharmacophore. Through a combination of molecular dynamic simulations and functional assays, we identify a putative binding site for GSK2820986A in the S4-S5 pocket and propose a potential inhibitory mechanism. Our findings provide a powerful tool compound and mechanistic framework to advance the investigation and therapeutic targeting of TRPC3 in disease.

biochemistry↗

bioRxiv2025

Large-Scale Structure-Based Virtual Screening Identifies Diverse KNa1.1 (KCNT1) Potassium Channel Inhibitors

Severe drug-resistant childhood epilepsy is caused by KCNT1 gain-of-function genetic variants, resulting in increased KNa1.1 channel activity. KCNT1-associated epilepsy is thought to affect around 1 in 300,000 births worldwide. Current treatment for KCNT1 epilepsy only provides mild symptomatic relief and uses a cocktail of experimental medications which must be personalised for the individual and are often poorly tolerated. Critically, with many patients, no therapeutic benefit is achieved. We sought to address this by using large-scale virtual screening to accelerate the development of a molecule which binds directly to KCNT1 to supress overactivity. We purchased a total of 71 compounds and using a combination of fluorescent thallium flux assays and patch clamp electrophysiology, identified a series of eight structurally diverse, novel inhibitors of the KNa1.1 channel with potency in the low micromolar range. These provide potential starting points for further development of drugs to treat KCNT1-associated epilepsy. HighlightsO_LIWe have discovered a range of structurally distinct inhibitors of the KCNT1 ion channel using large-scale virtual screening C_LIO_LIThese compound exhibit selectivity for the KCNT1 channel over other related ion channels C_LIO_LIThese compounds could provide starting points for new treatments for KCNT1 related Epilepsy. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/679465v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@481813org.highwire.dtl.DTLVardef@12494dforg.highwire.dtl.DTLVardef@a9f787org.highwire.dtl.DTLVardef@b9f4b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Identification of new KCNT1-epilepsy drugs by in silico, cell and Drosophila modelling.

ObjectiveHyperactive KCNT1 potassium channels, caused by gain-of-function mutations, are associated with a range of epilepsy disorders. Patients typically experience drug-resistant seizures and in cases with infantile onset, developmental regression can follow. KCNT1-related disorders include epilepsy of infancy with migrating focal seizures and sleep related hypermotor epilepsy. There are currently no effective treatments for KCNT1-epilepsies, but suppressing over-active channels poses a potential strategy. MethodsUsing KCNT1 channel structural data, we in silico screened a library of known drugs for those predicted to block the channel pore to reduce the current amplitude and inhibit channel activity. ResultsEight known drugs were investigated in vitro for their effects on patient-specific mutant KCNT1 channels, with four drugs showing significant reduction of K+ current amplitudes. The action of the four drugs was then analyzed in vivo and two were found to reduce the seizure phenotype in humanized Drosophila KCNT1-epilepsy models. InterpretationThis study identified two known drugs, antrafenine and nelfinavir mesylate, that reduce KCNT1 channel activity and reduce seizure activity in whole animals, suggesting their potential use as new treatments for KCNT1-epilepsy. The sequential in silico, in vitro and in vivo mechanism-based drug selection strategy used here may have broader application for other human disorders where a disease mechanism has been identified.

pharmacology and toxicology↗

bioRxiv2023

A molecular switch in RCK2 triggers sodium-dependent activation of KNa1.1 (KCNT1) potassium channels.

The Na+-activated K+ channel KNa1.1, encoded by the KCNT1 gene, is an important regulator of neuronal excitability. How intracellular Na+ ions bind and increase channel activity is not well understood. Analysis of KNa1.1 channel structures indicate that there is a large twisting of the {beta}N-Q loop in the intracellular RCK2 domain between the inactive and Na+-activated conformations, with a lysine (K885, human subunit numbering) close enough to form a salt bridge with aspartate (D839) in the Na+-activated state. Concurrently, an aspartate (D884) adjacent in the same loop adopts a position within 4 [A] of several acidic or polar residues. In carrying out mutagenesis and electrophysiology with human KNa1.1, we found alanine substitution of each of these residues resulted in almost negligible currents in the presence of up to 40 mM intracellular Na+. The exception was D884A, which resulted in constitutively active channels in both the presence and absence of intracellular Na+. Further mutagenesis of this site revealed an amino acid size-dependent effect. Substitutions at this site by an amino acid smaller than aspartate (D884V) also yielded constitutively active KNa1.1, D884I had Na+- dependence similar to wild-type KNa1.1, whilst increasing the side chain size larger than aspartate (D884E or D884F) yielded channels that could not be activated by up to 40 mM intracellular Na+. We conclude that Na+ binding results in a conformational change that accommodates D884 in the acid-rich pocket, which triggers further conformational changes in the RCK domains and channel activation. Statement of SignificanceSodium-activated potassium channels regulate neuronal excitability, and their dysfunction causes severe childhood disorders. Here, we identify a structural determinant in the intracellular domains that is responsible for triggering channel activation in response to sodium ion binding. An increase in the size of a particular amino acid renders the channel sodium-insensitive, whilst a decrease in size enables the channel to activate in the absence of sodium. This enhances our understanding of how this subclass of potassium channels respond to changes in the intracellular ionic environment. Furthermore, this may also further our understanding of the basis of human neurological disorders and their treatment.

biophysics↗

bioRxiv2021

Epilepsy-causing KCNT1 variants increase KNa1.1 channel activity by disrupting the activation gate.

Gain-of-function pathogenic missense KCNT1 variants are associated with several developmental and epileptic encephalopathies (DEE). With few exceptions, patients are heterozygous and there is a paucity of mechanistic information about how pathogenic variants increase KNa1.1 channel activity and the behaviour of heterotetrameric channels comprising both wild-type (WT) and variant subunits. To better understand these, we selected a range of variants across the DEE spectrum, involving mutations in different protein domains and studied their functional properties. Whole-cell electrophysiology was used to characterise homomeric and heteromeric KNa1.1 channel assemblies carrying DEE-causing variants in the presence and absence of 10 mM intracellular sodium. Voltage-dependent activation of homomeric variant KNa1.1 assemblies were more hyperpolarised than WT KNa1.1 and, unlike WT KNa1.1, exhibited voltage-dependent activation in the absence of intracellular sodium. Heteromeric channels formed by co-expression of WT and variant KNa1.1 had activation kinetics intermediate of homomeric WT and variant KNa1.1 channels, with residual sodium-independent activity. In general, WT and variant KNa1.1 activation followed a single exponential, with time constants unaffected by voltage or sodium. Mutating the threonine in the KNa1.1 selectivity filter disrupted voltage-dependent activation, but sodium-dependence remained intact. Our findings suggest that KNa1.1 gating involves a sodium-dependent activation gate that modulates a voltage-dependent selectivity filter gate. Collectively, all DEE-associated KNa1.1 mutations lowered the energetic barrier for sodium-dependent activation, but some also had direct effects on selectivity filter gating. Destabilisation of the inactivated unliganded channel conformation can explain how DEE-causing amino acid substitutions in diverse regions of the channel structure all cause gain-of-function.

neuroscience↗

bioRxiv2019

Structure-based identification and characterisation of novel inhibitors of KNa1.1 potassium channels, a stratified target for KCNT1-related epilepsy.

Several types of drug-resistant epileptic encephalopathies of infancy have been associated with mutations in the KCNT1 gene, which encodes the sodium-activated potassium channel subunit KNa1.1. These mutations are commonly gain-of-function, increasing channel activity, therefore inhibition by drugs is proposed as a stratified approach to treat disorders. To date, quinidine therapy has been trialled with several patients, but mostly with unsuccessful outcomes, which has been linked to its low potency and lack of specificity. Here we describe the use of a cryo-electron microscopy-derived KNa1.1 structure and mutational analysis to identify the quinidine biding site and identified novel inhibitors that target this site using computational methods. We describe six compounds that inhibit KNa1.1 channels with low- and sub-micromolar potencies, likely through binding in the intracellular pore vestibule. In preliminary hERG inhibition and cytotoxicity assays, two compounds showed little effect. These compounds may provide starting points for the development of novel pharmacophores for KNa1.1 inhibition, with the view to treating KCNT1-associated epilepsy and, with their potencies higher than quinidine, could become key tool compounds to further study this channel. Furthermore, this study illustrates the potential for utilising cryo-electron microscopy in ion channel drug discovery.

pharmacology and toxicology↗